This proposal is Project 1 within a P01 renewal, entitled Intracellular pathogens and innate immunity. Listeria monocytogenes is a highly amenable model intracellular pathogen that induces robust and long-lived cell mediated immunity. The goal of the previous proposal was to identify L. monocytogenes determinants that activate a host cytosolic surveillance pathway (CSP) of innate immunity that leads to the expression of IFNB and co-regulated genes. We hypothesize that this pathway contributes significantly to the potent and long-lived cell-mediated immunity induced by L. monocytogenes. Using a genetic screen and subsequent biochemistry, we identified c-di-AMP as the listerial ligand that is secreted through a multidrug efflux pump and activates the CSP. Thus, c-di-AMP is a newly discovered PAMP. C-di-AMP is also a novel and conserved small bacterial signaling molecule that appears essential for bacterial growth. We have identified the di-adenylate cyclase (DacA) and c-di-AMP phosphodiesterase (Pde). In Aim 1, we propose to further characterize the molecular determinants that lead to expression of c-di-AMP and generate a panel of well-characterized strains that express either enhanced or diminished levels of c-di-AMP. We will also address the following questions: What are the bacterial components that regulate c-di-AMP production? Does c-di- AMP act from the inside and/or the outside of bacteria? What is the physiologic role of c-di-AMP? In Aim 2, we will use the strains constructed above to determine the role of c-di-AMP during infection of macrophages and dendritic cells in vitro. We will also evaluate the effects of purified c-di-AMP on dendritic cell activation. In collaboration with the Vance Lab (Project 3), we will use ENU mutagenesis to identify novel host factors that control the CSP. In preliminary data, we identified Sting (Stimulator of interferon genes) as a critical host component necessary for response to L. monocytogenes, M. tuberculosis, and c-di-AMP. In the final Aim, we will test the hypothesis that c-di-AMP production is essential for the full expression of L. monocytogenes pathogenesis, and importantly, contributes to the induction of cell-mediated immunity. We will examine the role of c-di-AMP as an adjuvant.